DNA purification by solid phase extraction using a PCl3 modified glass fiber membrane

ABSTRACT

The present invention relates to modified glass fiber membranes which exhibit sufficient hydrophilicity and sufficient electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the membrane. Generally, the hydrophilic and electropositive characteristics are expressed at the surface of the modified glass fiber membrane. Preferred modified glass fiber membranes of the present invention include glass fiber membranes that have been modified by treatment with trifluoroacetic acid (TFA), BCl3, SiCl4, NaOH, F-, AlCl3 alone or in combination, with or without water. The modified glass fiber membranes of the present invention are particularly useful in processes for purification of DNA from other cellular components. In these processes, a suspension of cellular components is placed in contact with the modified glass fiber membrane, the modified glass fiber membrane is washed to remove all cellular components other than DNA which are bound to the membrane, and the bound DNA is eluted from the membrane.

BACKGROUND OF THE INVENTION

The present invention relates generally to the purification of DNA bysolid phase extraction, and more specifically to modified glass fibermembranes which are capable of binding DNA and eluting DNA undersuitable conditions.

The preparation of high-purity double-stranded (ds) plasmid DNA,single-stranded (ss) phage DNA, chromosomal DNA and agarose gel-purifiedDNA fragments is of critical importance in molecular biology. Ideally, amethod for purifying DNA should be simple, rapid and require little, ifany, additional sample manipulation. DNA rendered by such a methodshould be immediately amenable to transformation, restriction analysis,ligation or sequencing. A method with all of these features would beextremely attractive in the automation of DNA sample preparation, a goalof research and diagnostic laboratories. Typically, the preparation ofplasmid DNA from crude alcohol precipitates is laborious, most oftenutilizing CsCl gradients, gel filtration, ion exchange chromatography,or Rnase, proteinase K and repeated alcohol precipitation steps. Thesemethods also require considerable downstream sample preparation toremove CsCl and other salts, ethidium bromide and alcohol. Similararguments extend when using any of these methods for purifying DNAfragments. A further problem with these methods is that small,negatively-charged cellular components can co-purify with the DNA. Thus,the DNA can have an undesirable level of contamination.

DNA can also be purified using solid phases. Conventional solid phaseextraction techniques have utilized surfaces which either (1) fail toattract and hold sufficient quantities of DNA molecules because ofsurface design to permit easy recovery of the DNA molecules duringelution, or (2) excessively adhere DNA molecules to the surface, therebyhindering recovery of the DNA molecules during elution. Conventionalsurface materials which cause these problems when utilized in solidphase extraction include silica surfaces such as glass and Celite.Adequate binding of DNA to these types of surfaces can be achieved onlyby utilizing high concentrations of chaotropes or alcohols which aregenerally toxic, caustic, and/or expensive. For example, it is knownthat DNA will bind to crushed glass powders and to glass fiber filtersin the presence of chaotropes. The chaotropic ions typically are washedaway with alcohol, and the DNAs are eluted with low-salt solutions orwater. Importantly, RNA and protein do not bind. However, a seriousdrawback in the use of crushed glass powder is that its binding capacityis low. In addition, glass powders often suffer from inconsistentrecovery, incompatibility with borate buffers and a tendency to nicklarge DNAs. Similarly, glass fiber filters provide a nonporous surfacewith low DNA binding capacity. Other silicas, such as silica gel andglass beads, are not suitable for DNA binding and recovery. Currently,the solid phase of choice for solid phase extraction of DNA is Celitesuch as found in Prep-A-Gene™ by Bio-Rad Laboratories. As with thecrushed glass powders, high concentrations of chaotropes are requiredfor adequate binding of the DNA to the Celite.

SUMMARY OF THE INVENTION

These problems with conventional DNA purification methods are addressedby the present invention, which relates to modified glass fibermembranes which exhibit sufficient hydrophilicity and sufficientelectropositivity to bind DNA from a suspension containing DNA andpermit elution of the DNA from the material. Generally, the hydrophilicand electropositive characteristics are expressed at the surface of themodified glass fiber membrane, and are quantified as the presence ofoxygen as measured by Fourier transform infrared spectroscopy (FTIR) andthe presence of the substituted atom as detected by electron surfacecomposition analysis (ESCA). Preferred modified glass fiber membranes ofthe present invention include glass fiber membranes that have beenmodified by treatment with trifluoroacetic acid (TFA), BCl₃, SiCl₄,NaOH, F⁻, AlCl₃ alone or in combination, and further with or withouttreatment with water.

The modified glass fiber membranes of the present invention areparticularly useful in processes for purification of DNA from othercellular components. In these processes, a suspension of cellularcomponents is placed in contact with the modified glass fiber membrane,the modified glass fiber membrane is washed to remove all cellularcomponents other than DNA which are bound to the membrane, and the boundDNA is eluted from the membrane.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a modified glass fiber membrane whichexhibits sufficient hydrophilicity and sufficient electropositivity tobind DNA from a suspension of cellular components and permit elution ofthe DNA from the membrane. It has been found that much lowerconcentrations of chaotropes or alcohols can be utilized to achievepurification of DNA using the instant modified glass fiber membranes.Binding of DNA to the modified glass fiber membranes allows the use ofsuch membranes for automated DNA isolation processes and for sampleprocessing steps for DNA probe tests. For example, using a dot-blotapparatus, 96 samples can be done at once, and rapidly.

DNA interacts with a solid phase surface in two ways. First, DNAinteracts with the surface through hydrogen bonding between hydroxylgroups of DNA and surface components of the solid phase, such as surfacehydroxyls. The second interaction is between the 10 negatively chargedphosphates of the DNA and positively charged elements of the solid phasesurface. The hydrophilic and electropositive characteristics of thesolid phase surface must be such as to allow binding of the DNA from asuspension of cellular components, a suspension of nucleic acids andother materials and/or a suspension of nucleic acids, and to permitelution of the DNA from the solid phase material. Thus, theelectropositive characteristics of the solid phase material cannot havetoo high of a positive charge, or the DNA will stick to the surface andcannot be eluted. This characteristic is also true for many metal-basedsurfaces, which has resulted in their inability to be utilized forpurification of DNA.

Silicon-containing materials, e.g., silica, Celite, glass powders andthe like, have been used for DNA purification with mixed results. Someof these surfaces have low binding capacities and/or require the use ofhighly concentrated solutions of chaotropes or alcohols for the bindingof DNA. Thus, it is desired to produce solid phase surfaces,particularly solid phases of modified glass fiber membranes, whichexhibit suitable hydrophilic and electropositive characteristics for DNApurification and/or for DNA purification with much lower concentrationsof chaotropes or alcohols. On the surface of the solid phase,hydrophilic characteristics are achieved by the presence of groups thatwill attract water molecules. Suitable groups include --OH, --NH, --F,--H or groups with double-bonded oxygen such as carbonyl, sulfonyl orphosphonyl. Electropositive characteristics are achieved by the presenceof positively charged atoms. Suitable positively-charged atoms includeSi, B or Al. In accordance with the present invention, modified glassfiber membranes are prepared in which the hydrophilic characteristicsare achieved by incorporation of the appropriate hydrophilic groups, andthe electropositive characteristics are achieved by incorporation of Siand other appropriate positively-charged atoms. Preferred modified glassfiber membranes of the present invention include glass fiber membranesthat have been modified by treatment with trifluoroacetic acid (TFA),BCl₃, SiCl₄, NaOH, F⁻, AlCl₃, alone or in combination, and further withor without treatment with water.

In general, the modified glass fiber membranes of the present inventionare prepared by treating a glass fiber membrane with either TFA or 0.2NNaOH overnight at 15° C. to 30° C., preferably at 20° C. to 25° C., andmost preferably at room temperature. The TFA- or NaOH-treated glassfiber membranes could be used directly or could be further treated withNaOH, BCl₃, SiCl₄, AlCl₃ or F⁻, and if desired, further treated with0.2N NaOH or H₂ O. These latter treatments were generally conducted aspreviously described except the treatment with AlCl₃ and SiCl₄ was mostpreferably at 22° C. to 35° C. Several of the modified glass fibermembranes gave superior performance (i.e., a greater recovery of DNA)when compared to the unmodified surfaces, and others provided the samerecovery of DNA. Glass fiber membranes which were modified such thatundesired hydrophilicity and/or electropositivity resulted, provided norecovery of DNA. Glass fiber membranes, such as Whatman G/F B or Cmembranes, treated with the following materials, provided recovery ofDNA:

TFA

TFA, BCl₃ and H₂ O

NaOH, SiCl₄ and NaOH

NaOH, BCl₃ and H₂ O

NaOH, PCl₃ and H₂ O

NaOH

TFA and NaOH

TFA and F⁻

TFA and BCl₃

NaOH, BCl₃ and NaOH

NaOH, AlCl₃ and NaOH

Glass fiber membranes, such as Whatman G/F B or C membranes, treatedwith the following materials, provided no recovery of DNA:

TFA, AlCl₃ and H₂ O

TFA, SiCl₄ and H₂ O

TFA and SiCl₄

TFA and AlCl₃

NaOH, AlCl₃ and H₂ O

NaOH, SiCl₄ and H₂ O

The modified glass fiber membranes of the present invention are used forthe purification of DNA from other cellular components or potentialcontaminants. The DNA can be obtained from any source, including but notlimited to crude cell extracts, biological fluids, phage supernatants,agarose gels and radiolabelling reactions. The DNA can bedouble-stranded, single-stranded, circular or linear, and can bevariable in size. Conventional techniques for obtaining DNA from anysource, well known in the art, are utilized to prepare the DNA forpurification. Typical procedures for obtaining DNA end with a suspensionof the DNA in solution. For isolation of DNA from biological samples,see, e.g., Harding, J. D. et al., Nucleic Acids Research 17:6947 (1989)and Marko, M. A. et al., Analytical Biochemistry 121:382 (1982).Procedures for isolation of plasmid DNA can be found in Lutze, L. H. etal., Nucleic Acids Research 20:6150 (1990). Extraction ofdouble-stranded DNA from biological samples can be found in Yamada, O.et al., Journal of Virological Methods 27:203 (1990). Most DNA solutionscomprise the DNA in a suitable buffer such as TE (Tris-EDTA), TEA (40 mmTris-acetate, 1 mm EDTA) buffer, or a lysate. See also Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, New York (1989).

Once the DNA is obtained in a suitable solution or suspension, themodified glass fiber membrane of the present invention is added to thesolution or suspension. Alternatively, the DNA solution or suspensioncould be added to the modified glass fiber membrane of the presentinvention using a dot blot apparatus that pulls the suspension throughthe filter. After the DNA solution or suspension is contacted with themodified glass fiber membrane of the present invention, a binding buffertypically is added to assist in the binding of the DNA to the membrane.Suitable binding buffers include well-known chaotropes such as NaClO₄and NaI, and other agents such as guanidine Hcl, NaCl or isopropanol.After the DNA is bound to the modified glass fiber membrane, the pureDNA is eluted from the membrane. Suitable eluting agents include 10 mMTris, Ph 7.0 or water. Generally, the modified glass fiber membrane withbound DNA is washed prior to eluting the DNA. Suitable washing agentsinclude 80/20 ethanol/50 mM Tris, Ph 7.0 and other low molecular wightalcohols.

The DNA obtained by purification with the modified glass fiber membranesof the present invention may be used without further manipulation forrestriction enzyme digestion, cloning, sequencing, diagnostics and thelike. The high quality of DNA prepared with the present invention andthe speed with which DNA is purified, with minimal downstreamprocessing, mean that these modified glass fiber membranes can be usefulin the automation of DNA sample preparation.

The modified glass fiber membranes of the present invention allow veryquick and efficient isolation of DNA from biological samples. They cansubstantially decrease the time required to process pure DNA frombiological samples, compared with currently used techniques, and in somecases generate high quantities of pure DNA. With these advantages, themodified glass fiber membranes are useful for part of the sampleprocessing step in DNA probe tests. One example is use of the modifiedglass fiber membranes in a dot blot apparatus which allows the rapidprocessing of 96 samples at one time.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1 Synthesis of Modified Glass Fiber Membranes A. Treatment withTFA

Glass fiber membranes, Whatman G/F B or C, were treated with TFA(Aldrich Chemical Co.) by adding 20 ml of TFA to a 50 ml beaker. Themembranes were added to the beaker one at a time. The beaker with TFAand the membranes was allowed to stand overnight at room temperature.The TFA-treated membranes were then placed in a desiccator, attached toan aspirator with a KOH trap and stored under reduced pressure overnightto remove TFA.

B. Treatment with NaOH

Glass fiber membranes, Whatman G/F B or C, were treated with 0.2N NaOHby adding 20 ml of NaOH to a 50 ml beaker. The membranes were added tothe beaker one at a time. The beaker with NaOH and the membranes washeated on low (35° C.) for two hours, and then allowed to standovernight at room temperature. The NaOH-treated membranes were washedfour times with 20 ml water and three times with 20 ml acetone. TheNaOH-treated membranes were air-dried for 20 minutes, oven-dried onehour at 100° C., and stored in a desiccator.

TFA-treated glass fiber membranes prepared as described above were alsotreated with 0.2N NaOH, as described above, except that the TFA-treatedmembranes were added to 40 ml NaOH and heated on low (35° C.) overnight.

SiCl₄ -, BCl₃ - or AlCl₃ -treated--NaOH treated glass fiber membranesprepared as described below were treated with 0.2N NaOH to replacechlorine groups with hydroxyl groups, by adding 40 ml of NaOH to a 100ml beaker. The membranes were added to each beaker and heated on low(35° C.) overnight. The NaOH-treated membranes were filtered, placed ina fritted funnel, washed three times with 2 ml water and three timeswith 2 ml acetone, air-dried for 15 minutes, and oven-dried for one hourat 35° C. The NaOH-treated SiCl₄ -treated-NaOH-treated (hereinafterNaOH/SiCl₄ /NaOH-treated) glass fiber membranes, the NaOH-treated BCl₃-treated-NaOH-treated (hereinafter NaOH/BCl₃ /NaOH-treated) glass fibermembranes, and the NaOH-treated AlCl₃ -treated-NaOH-treated (hereinafterNaOH/AlCl₃ /NaOH-treated) glass fiber membranes were stored in adesiccator.

C. Treatment with Fluoride

TFA-treated glass fiber membranes prepared as described above weretreated with fluorine by adding (a) 5 ml of tetrabutylammonium fluoride(TBAF) and 5 ml tetrahydrofuran (THF) (5 mM), or (b) 10 ml TBAF (10 Mm)to 50 ml beakers. TFA-treated glass fiber membranes were added to eachbeaker and heated on low (35° C.) overnight. The fluoride-treatedmembranes were put in a fritted funnel and washed repeatedly with waterand then with acetone, air-dried ˜30 minutes, and oven-dried at 35° C.for one hour. The F⁻ -treated-TFA-treated (hereinafter TFA/F⁻ -treated)glass fiber membranes were stored in a desiccator.

D. Treatment with SiCl₄, BCl₃, AlCl₃ or PCl₃

TFA-treated glass fiber membranes prepared as described above weretreated with SiCl₄, BCl₃ or AlCl₃ by adding (a) 10 ml of BCl₃ (1M in CH₂; Aldrich Chemical Co.) and 5 ml of THF; (b) 10 ml of AlCl₃ (1M innitrobenzene; Aldrich Chemical Co.), or (c) 20 ml SiCl₄ (PetrachSystems) to 50 ml beakers. TFA-treated glass fiber membranes were addedto each beaker and heated on low (35° C.) overnight. The SiCl₄ -, BCl₃ -or AlCl₃ -treated membranes were washed repeatedly with acetone,air-dried ˜30 minutes, and oven-dried one hour at 35° C. The SiCl₄-treated-TFA-Treated (hereinafter TFA/SiCl₄ -treated) glass fibermembranes, AlCl₃ -treated-TFA-treated (hereinafter TFA/AlCl₃ -treated)glass fiber membranes, and BCl₃ -treated-TFA-treated (hereinafterTFA/BCl₃ -treated) glass fiber membranes were stored in a desiccator.

NaOH-treated glass fiber membranes prepared as described above weretreated with SiCl₄, BCl₃ or PCl₃ by adding (a) 20 ml SiCl₄ (PetrachSystems); (b) 20 ml BCl₃ (1N in CH₂ Cl₂ ; Aldrich Chemical Co.); (c) 20ml AlCl₃ (1M in nitrobenzene; Aldrich Chemical Co.); or (d) 20 ml PCl₃(2M in CH₂ Cl₂ ; Aldrich Chemical Co.) to a 100 ml beaker. NaOH-treatedglass fiber membranes were added to each beaker. Beakers containing themembranes and BCl₃, AlCl₃ or SiCl₄ were heated on low (35° C.)overnight. The beakers containing the membranes and BCl₃ or PCl₃ wereallowed to stand overnight at room temperature. The SiCl₄ -, BCl₃ -,AlCl₃ - or PCl₃ -treated membranes were washed alternatively either (a)three times with 10 ml water and three times with 10 ml acetone, or (b)three times with 10 ml acetone, three times with 10 ml water and threetimes with 10 ml acetone, air-dried and then oven-dried one hour at 35°C. The SiCl₄ -treated-NaOH-treated (hereinafter NaOH/SiCl₄ -treated)glass fiber membranes, BCl₃ -treated-NaOH-treated (hereinafter NaOH/BCl₃-treated) glass fiber membranes, and AlCl₃ -treated-NaOH-treated(hereinafter NaOH/AlCl₃ -treated) glass fiber membranes were stored in adesiccator.

E. Treatment with Water

The TFA/SiCl₄ -, TFA/BCl₃ -, TFA/AlCl₃ -, NaOH/SiCl₄ -, NaOH/BCl₃ -,NaOH/AlCl₃ - or NaOH/PCl₃ -treated glass fiber membranes prepared asdescribed above were treated with water to replace chlorine groups withhydroxyl groups, by adding 20 ml of water to a 50 ml beaker. Themodified glass fiber membranes were added to separate beakers and heatedon low (35° C.) overnight for the TFA-treated class or stored overnightat room temperature for the NaOH-treated class. The TFA-treated classwere then placed in a fritted funnel and washed three times with 15 mlacetone, air-dried for 15 minutes, and oven-dried one hour at 35° C. TheNaOH-treated class were washed three times with 10 ml water. The H₂O-treated-TFA/SiCl₄ -treated (hereinafter TFA/SiCl₄ /H₂ O -treated)glass fiber membranes, the H₂ O-treated-TFA/BCl₃ -treated (hereinafterTFA/BCl₃ /H₂ O -treated) glass fiber membranes, the H₂O-treated-TFA/AlCl₃ -treated (hereinafter TFA/AlCl₃ /H₂ O-treated) glassfiber membranes, the H₂ O-treated-NaOH/SiCl₂ -treated (hereinafterNaOH/SiCl₄ /H₂ O-treated) glass fiber membranes, the H₂O-treated-NaOH/BCl₃ -treated (hereinafter NaOH/BCl₃ /H₂ O-treated) glassfiber membranes, the H₂ O-treated-NaOH/AlCl₃ -treated (hereinafterNaOH/AlCl₃ /H₂ O-treated) glass fiber membranes, and the H₂O-treated-NaOH/PCl₃ -treated (hereinafter NaOH/PCl₃ /H₂ O-treated) glassfiber membranes were stored in a desiccator.

EXAMPLE 2 Analysis of DNA Recovery Using Modified Glass Fiber Membranes

A Bio-Rad Bio-Dot apparatus was used to test the membranes. A smallstrip of each membrane of the following membranes was cut large enoughto cover one well or two wells of the apparatus:

    ______________________________________                                                  Whatman G/F                                                         Membrane  Glass Fiber                                                         Number    Membrane       Modification                                         ______________________________________                                        1         B              NaOH                                                 2         C              TFA                                                  3         C              TFA/NaOH                                             4         C              TFA/F.sup.- (5 mM)                                   5         C              TFA/F.sup.- (10 mM)                                  6         C              TFA/AlCl.sub.3 /H.sub.2 O                            7         C              TFA/SiCl.sub.4 /H.sub.2 O                            8         C              TFA/BCl.sub.3 /H.sub.2 O                             9         C              TFA/BCl.sub.3                                        10        C              TFA/SiCl.sub.4                                       11        C              TFA/AlCl.sub.3                                       12        C              NaOH/SiCl.sub.4 /NaOH                                13        C              NaOH/BCl.sub.3 /NaOH                                 14        C              NaOH/AlCl.sub.3 /NaOH                                15        B              NaOH/AlCl.sub.3 /H.sub.2 O                           16        B              NaOH/BCl.sub.3 /H.sub.2 O                            17        B              NaOH/PCl.sub.3 /H.sub.2 O                            18        B              NaOH/SiCl.sub.4 /H.sub.2 O                           ______________________________________                                         (untreated membranes were used as controls)                              

After the membranes were properly aligned within the box and it wasclamped tightly, the unused wells were covered with tape to maintainvacuum pressure.

Each well was provided with 0.7 ml 6M NaCl₄ and 0.78 μg λ DNA (BRLCatalog No. 56125A). Water aspiration was used to apply gentle vacuum toavoid breakage of membranes. After the DNA/chaotrope solution was pulledthrough, the membranes were washed twice with 350 μl 80% ethanol/50 μMTRIS, pH 7.0. Membranes 7, 10 and 18 did not allow flow-through of DNAchaotrope solution, which was then manually suctioned off prior towashing. The membranes were then removed from the apparatus and trimmedto the shape of the well (which is imprinted on the fitter) and placedin a 0.65 ml microcentrifuge tube. Thirty-five μl of elution buffer(TE=10 mM Tris, 1 mM EDTA, pH 8.0) was added to each tube and incubatedfor 10 minutes at 40° C. The samples were then spun for about threeminutes. Twenty μl of buffer was placed in another tube and about 1 μlType II Loading Dye (25% Ficoll, 0.25% Bromophenol Blue, 0.25% xyleneglycol) was added. Gel electrophoresis was performed on 1% agarose, 1XTAE gel for 25 minutes at 130 volts. The gels were stained in ethidiumbromide in water (1:100 dilution of 10 mg/ml stock) for 10 minutes anddestained for 15 minutes. Photographs were taken over UV light with Type57 Polaroid film.

Membranes 2, 8, 12, 16 and 17 recovered DNA better than all of the othermembranes, including the unmodified membrane controls. Membrane 17appeared to recover close to 100% of the DNA. Membranes 1, 3, 4, 5, 9,13 and 14 recovered DNA apparently about the same as the unmodifiedcontrols. Membranes 6, 7, 10, 11, 15 and 18 did not provide any recoveryof DNA.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

What is claimed is:
 1. A modified glass fiber membrane which exhibitssufficient hydrophilicity and sufficient electropositivity to bind DNAfrom a suspension containing DNA and permit elution of the DNA from themembrane, said glass fiber membrane treated first with NaOH, then withPCl₃ and finally with H₂ O.
 2. A method for purifying DNA comprising thesteps of:(a) contacting a suspension containing DNA with the modifiedglass fiber membrane of claim 1 under conditions suitable to bind DNA tosaid membrane; (b) washing said membrane having bound DNA; and (c)eluting the DNA from said membrane.